The apical surface of mammalian urothelium is almost completely covered by rigid-looking urothelial plaques consisting of 16 nm uroplakin particles that are hexagonally packed into two-dimensional crystals. Highly purified urothelial plaques are comprised of four major uroplakins (UP), i.e., uroplakin Ia (27 kDa), Ib (28 kDa), II (15 kDa) and III (47 kDa). The two tetraspanin members UPIa and UPIb interact preferentially with the two single transmembrane-domained uroplakins II and III, respectively, forming two uroplakin pairs consisting of UPIa/II and UPIb/III. During the last grant period, we genetically ablated mouse genes encoding for uroplakins II and III. Mice deficient in these two major urothelial differentiation products have a grossly abnormal urothelium lacking typical umbrella cells; have abnormally small urothelial plaques; and suffer from vesicoureteral reflux, hydronephrosis and, in some cases, neonatal death. These results indicate that uroplakins are integral subunits of the urothelial plaques which contribute to the permeability barrier function, and that urothelial defects may play a role in certain urinary tract diseases. Little is known, however, about how uroplakin defects may affect urothelial and bladder function as a whole, how uroplakins interact with cytoplasmic and other membrane-associated proteins, and how surface-accessible are the uroplakin receptors to the uropathogenic bacteria. The proposed studies in this Project will attempt to answer some of these questions, by studying how the ablation of uroptakin genes affects the permeability barrier and micturition functions of the bladder (Aim 1); how uroplakins interact with some cytoplasmic proteins that may play a role in mediating uroplaldn:cytosketetal interaction and signal transduction (Aim 2); and how accessible uroplakin molecules are on the apical surface of urothelial umbrella cells, and whether there are other uroplakin-associated cell surface proteins that may be related to the urotheliat 'glycocalyx' coating that has been hypothesized to play a key role in several bladder diseases (Aim 3). Results from this Project will shed light on the structure, function and regulation of urothelial plaques, and will have major implications on the molecular etiology and pathogenesis of several important bladder diseases including interstitial cystitis, urinary tract infection and bladder cancer formation.